1. Field of the Invention
The present invention relates to agents for use in a method of detection of 8-oxoguanine, 8-oxodeoxyguanosine, 8-oxoadenine and 8-oxodeoxyadenosine, in particular for the detection of damaged nucleic acids, diagnostic tests for same and methods for the detection of same.
2. Description of the Related Art
Nucleic acid base damage, in particular oxidative base-damage of DNA, may arise as a result of a number of causes including toxins, carcinogenesis and neurodegenerative disorders. The detection of such damage is important in many fields of medicine including medical diagnostics, pathology and occupational health.
Present techniques for the detection of nucleic acid base damage rely on detection of 8-oxoguanine, a sensitive marker of DNA base-damage caused by oxygen free radicals (Kasai, H. and Nishimura, S., In: Sies, H., ed. Oxidative Stress: Oxidants and Antioxidants. London: Academic Press, 1991:99-116; Ames, B. N., 1989, Free Rad. Res. Comm., 7: 121-128), although 8-oxoguanine is only one of at least 20 products formed.
The use of 8-oxoguanine as a marker of oxidative damage to DNA followed the description of its analysis as the deoxynucleoside (8-oxodeoxyguanosine) in DNA digests by HPLC (high pressure liquid chromatography) with electrochemical detection (Floyd, R. A. et al., 1986, Free Rad. Res. Commun. 1: 163-172). Alternatively, gas chromatography-mass spectrometry (GC-MS) methods have been used to quantitate 8-oxoguanine as the free base (Dizdaroglu, M., 1994, Methods Enzymol., 234: 3-16), but the technique is expensive and technically demanding to use. Nevertheless the two techniques have been compared (as reviewed in Halliwell, B. and Dizdaroglu, M., 1992. Free Rad. Res. Commun. 16: 75-87) and results obtained do not concur. In general. levels of 8-oxoguanine are higher when determined by the GC-MS technique and even in freshly isolated cells, 2-11 fold higher values have been reported by GC-MS.
A number of other approaches to the determination of 8-oxoguanine in DNA have been described. .sup.32 P-postlabelling procedures are well established in the literature (Lu, L. J. W. et al., 1991, Chem. Pharmaceut. Bull. 39: 1880-1882; Povey, A. C. et al., In: Postlabelling Methods for Detection of DNA Adducts. Lyon, International Agency for Research on Cancer, 1993:105-114); these methods provide the potential for very sensitive detection although the techniques are very time-consuming and cumbersome. Capillary electrophoretic determinations of 8-oxodeoxyguanosine (Guarnieri C. et al., 1994, J. Chromatogr. B. 656: 209-213) and 8-oxoguanine (Poon, K. W. et al., 1995, Biochem. Soc. Trans. 23: 433s) have been described. To date, these techniques lack concentration sensitivity due to inherent lack of sensitivity of UV absorbence measurements.